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Bmal1- and Per2-mediated regulation of the osteogenic differentiation and proliferation of mouse bone marrow mesenchymal stem cells by modulating the Wnt/β-catenin pathway

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Abstract

Background

Bmal1 and Per2 are the core components of the circadian clock genes (CCGs). Bmal1−/− mice exhibit premature aging, as indicated by hypotrichosis and osteoporosis, with a loss of proliferation ability. The same occurs in Per2−/− mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. Thus, our study aimed to explore the effects and specific mechanism.

Methods and results

Lentiviral and adenoviral vectors were constructed to silence or overexpress Bmal1 or Per2 and MTT, flow cytometry, RT-qPCR, WB, immunohistochemistry, alizarin red staining and ChIP-Seq analyses were applied to identify the possible mechanism. The successful knockdown and overexpression of Bmal1/Per2 were detected by fluorescence microcopy. Flow cytometry found out that Bmal1 or Per2 knockdown resulted in G1-phase cell cycle arrest. RT-qPCR showed the different expression levels of Wnt-3a, c-myc1 and axin2 in the Wnt/β-catenin signaling pathway as well as the gene expression change of Rorα and Rev-erbα. Meanwhile, related proteins such as β-catenin, TCF-1, and P-GSK-3β were detected. ALP activity and the amount of mineral nodules were compared. ChIP-Seq results showed the possible mechanism.

Conclusions

Bmal1 and Per2, as primary canonical clock genes, showed synergistic effects on the proliferation and differentiation of BMSCs. They would inhibit the Wnt/β-catenin signaling pathway by downregulating Rorα expression or upregulating Rev-erbα expression, both of which were also key elements of CCGs. And this may be the mechanism by which they negatively regulate the osteogenic differentiation of BMSCs.

Graphical abstract

Bmal1 and Per2 show synergistic effects in the proliferation of BMSCs. In addition, they play a synergistic role in negatively regulating the osteogenic differentiation ability of BMSCs. Bmal1 and Per2 may regulate the aging of BMSCs by altering cell proliferation and osteogenic differentiation through Rorα and Rev-erbα to affect Wnt/β-catenin pathway.

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Data availability

All datasets generated and/or analyzed during this study are included in this published article.

Abbreviations

BMSCs:

Bone marrow mesenchymal stem

Bmal1:

Brain and muscle arant-like-1

Per2:

Period 2

Clock:

Circadian locomotor output cycles kaput

Cry:

Cryptochrome

RORα:

Retinoic acid-related orphan

Rev-erb:

Retinoid-related orphan receptor α

PCR:

Polymerase chain reaction

RT-PCR:

Reverse transcription polymerase chain

RT-qPCR:

Real-time fluorescence quantitative

FBS:

Fetal bovine serum

PBS:

Phosphate-buffered saline

DMSO:

Dimethyl sulfoxide

EDTA:

Ethylene diamine tetraacetic acid

FCM:

Flow cytometry

WB:

Western blot

SA-β-gal:

Senescence-associated β-galactosidase

GFP:

Green fluorescent protein

RFP:

Red fluorescent protein

BSA:

Bovine serum albumin

MTT:

Methyl thiazolyl tetrazolium

CCGs:

Circadian clock genes

ALP:

Alkaline phosphatase

OCN:

Osteocalcin

Runx2:

Runt-related transcription factor 2

Wnt3a:

Wingless Int1 3a

C-myc:

Cell-myc1

TCF1:

T-cell-specific transcription factor1

P-GSK-3:

Phosphorylated glycogen synthase kinase-3β

ChIP-Seq:

Chromatin immunoprecipitation and high-throughput sequencing

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Funding

This work was supported by the National Science Foundation of China (No. 81870745 and No. 81371113).

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Author notes

  1. Jiawen Zheng and Lanxin Zhang have contributed equally to this work.

Authors and Affiliations

  1. Orthodontic Centre, West China College of Stomatology, Sichuan University, Chengdu, China

    Jiawen Zheng, Lanxin Zhang, Qing Zhao, Xiaoyu Wei, Yuqing Yang & Rong Li

  2. State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China

    Jiawen Zheng, Lanxin Zhang, Zhen Tan, Qing Zhao, Xiaoyu Wei, Yuqing Yang & Rong Li

  3. Oral Implant Centre, West China Hospital of Stomatology, Sichuan University, Chengdu, China

    Zhen Tan

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  1. Jiawen Zheng
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  4. Qing Zhao
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Contributions

JZ and LZ designed the projects, acquired the data and wrote the manuscript. QZ and ZT designed the projects and approved the draft. XW and YY constructed the figures and approved the draft. RL interpreted the data and approved the draft.

Corresponding authors

Correspondence to Zhen Tan or Qing Zhao.

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The authors declare that they have no conflict of interest.

Ethical approval and consent to participate

The Research Ethics Committee of State Key Laboratory of Oral Diseases reviewed our research project and made the resolution as the following: The experiment involved ethical part conforms to the scientific experiment ethical requirement, it is agreed to implement the experiment.

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Zheng, J., Zhang, L., Tan, Z. et al. Bmal1- and Per2-mediated regulation of the osteogenic differentiation and proliferation of mouse bone marrow mesenchymal stem cells by modulating the Wnt/β-catenin pathway. Mol Biol Rep 49, 4485–4501 (2022). https://doi.org/10.1007/s11033-022-07292-6

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